A method for preserving Dunaliella salina algal species with high salinity in a green bottle

Abstract

The present invention discloses a preservation nutrient solution for Dunaliella salina and a method for preserving Dunaliella salina algal species with a green bottle at high salinity, which relates to the technical field of methods for preserving Dunaliella salina algal species. The present invention uses a green bottle to preserve Dunaliella salina algal species, including the following steps: (1) adding the preservation nutrient solution into the green bottle and inoculating the Dunaliella salina algal species; (2) storing in a dark and low-temperature environment for 48 h, where the low temperature is 0-2 °C; (3) transferring to a light incubator for preservation, with a light intensity of 600-900 lux, a light-dark cycle of L / D = 6 h / 18 h, and a temperature of 4-9 °C. Compared with the prior art, the method for preserving Dunaliella salina with the preservation nutrient solution for Dunaliella salina and the green bottle at high salinity of the present invention significantly prolongs the preservation time and survival rate of Dunaliella salina, reduces the cost of subculture preservation of Dunaliella salina, and can continuously and stably supply Dunaliella salina algal species to the market.

Description

Technical Field

[0001] The present invention relates to the technical field of the preservation method of Dunaliella salina strains, and more specifically, to a method for preserving Dunaliella salina strains with a high salinity in a green bottle. Background Art

[0002] Dunaliella salina, also known as saltwater dunaliella, is a green alga that can live in high-salinity seawater and often turns red in a high-salt environment. The most prominent feature of Dunaliella salina is that it contains a large amount of β-carotene, the content of which is many times higher than that of β-carotene in carrots and fruits, and it has broad market prospects.

[0003] Currently, the preservation techniques of microalgae (including Dunaliella salina) mainly include: subculture preservation method, drying preservation method, immobilization preservation method, cryopreservation method, etc. The subculture preservation method is the commonly used method at present. The disadvantage of this method is that the nutrient salts are consumed quickly, frequent subcultures are required, the workload is large, and contamination is likely to occur, and it can only be used for the short-term preservation of algae. CN105886403A discloses a method for preserving microalgae strains for medium and long-term, but the preservation effects of this method on different species of algae are different and it is not applicable to the preservation of Dunaliella salina.

[0004] Therefore, how to provide a method suitable for the medium and long-term preservation of Dunaliella salina strains is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for preserving Dunaliella salina strains with a high salinity in a green bottle.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A nutrient solution for preserving Dunaliella salina strains comprises the following raw materials in parts by weight: 90 - 95 parts of magnetized and disinfected seawater, 3 - 7 parts of beer, 12 - 15 parts of NaCl, 0.03 - 0.07 parts of NH4NO3, 0.0004 - 0.0008 parts of KH2PO4, 0.00005 - 0.00009 parts of ferric citrate, 0.3 - 0.8 parts of NaHCO3, 0.02 - 0.05 parts of glycerol, 0.000003 - 0.000007 parts of kalamycin, and 0.0000006 - 0.0000009 parts of choline chloride.

[0008] The magnetization and disinfection of seawater in the present invention helps to increase the activity and density of water molecules in the water body, provides a stable preservation environment for Dunaliella salina, and can improve the self-recovery ability of Dunaliella salina after magnetization, and has bactericidal and disinfection effects. Beer is rich in nutrients, and the carbohydrates, CO2, alcohols, and organic acids it contains can, together with "NH4NO3, KH2PO4, ferric citrate, NaHCO3", etc., provide a carbon source and energy substances for the growth of Dunaliella salina, improve the preservation environment of Dunaliella salina, and significantly extend the preservation period; the CO2 in beer can also stabilize the pH value of the culture solution during the preservation process, which is beneficial for preservation; and beer also has a certain bactericidal effect. The content of NaCl in the nutrient preservation solution of the present invention is 120-150‰, which is 3-5 times that of ordinary seawater, effectively suppressing the reproduction of microorganisms such as bacteria, other microalgae, and viruses. However, since Dunaliella salina is tolerant to high salinity, this concentration range will not affect the survival of Dunaliella salina. Glycerol is used as an antifreeze to prevent Dunaliella salina from being frozen at low temperatures. Kanamycin is a bactericide that inhibits the reproduction of miscellaneous bacteria and viruses; choline chloride is a plant growth regulator that is beneficial to the resistance of Dunaliella salina.

[0009] As a preferred technical solution, the nutrient preservation solution for Dunaliella salina includes the following raw materials in parts by weight: 92 parts of magnetized and disinfected seawater, 4 parts of beer, 13 parts of NaCl, 0.05 part of NH4NO3, 0.0005 part of KH2PO4, 0.00006 part of ferric citrate, 0.6 part of NaHCO3, 0.03 part of glycerol, 0.000005 part of kanamycin, and 0.0000007 part of choline chloride.

[0010] The preparation method of the magnetized and disinfected seawater is as follows: ordinary seawater is filtered through absorbent cotton, boiled, cooled, and then flows through a water magnetizer (magnetic induction intensity is 0.3T, flow rate is 3t / h, flow velocity is 0.2m / s) to obtain it.

[0011] As a preferred technical solution, the NaCl is added in three equal amounts. The first addition is before inoculation, and the second and third additions are 12 hours and 24 hours after inoculation respectively.

[0012] Another object of the present invention is to provide a method for preserving Dunaliella salina algal seeds in a green bottle with high salinity. The method for preserving Dunaliella salina algal seeds in a green bottle includes the following steps:

[0013] (1) Add the above-mentioned preservation nutrient solution to the green bottle body and inoculate the Dunaliella salina algal seeds. The addition amount of the preservation nutrient solution is 1 / 3 to 1 / 2 of the volume of the bottle body;

[0014] (2) Place it in a dark and low-temperature environment for 48h, and the low temperature is 0-2°C;

[0015] (3) Transfer it to a light incubator for preservation. The light intensity is 600 - 900 lux, the light - dark cycle is L / D = 6 h / 18 h, and the temperature is 4 - 9 °C.

[0016] The method of the present invention uses a green bottle body to preserve Dunaliella salina. Dunaliella salina is a kind of green alga that prefers green light. When natural light passes through the green bottle body, it becomes dim green light, enabling Dunaliella salina to not only grow but also maintain a relatively low metabolic level, prolong its lifespan, and is conducive to the preservation of Dunaliella salina. After inoculating the Dunaliella salina strain into the preservation nutrient solution, it is first placed in a low - temperature environment of 0 - 2 °C for 48 h to adapt it to the light - free and low - temperature environment, and then transferred to a long - term preservation in a low - temperature and low - light environment, which can significantly improve the preservation survival rate. The method of the present invention has a long preservation time, a high survival rate, simple equipment, easy operation, is not easily contaminated, has a low cost, and a small workload.

[0017] As a preferred technical solution, the thickness of the green bottle body ≥ 1.5 mm.

[0018] More preferably, the green bottle is a beer bottle.

[0019] As a preferred technical solution, the step of inoculating the Dunaliella salina strain in step (1) is: taking a pure Dunaliella salina strain in the exponential growth phase for inoculation, with an inoculation density of 10,000 - 30,000 cells / ml, gently shaking it evenly, with a shaking time of 25 - 30 seconds, and then plugging the bottle mouth with a breathable sterile plug.

[0020] As a preferred technical solution, the breathable sterile plug is a plug made of medical gauze wrapped around absorbent cotton.

[0021] As a preferred technical solution, in step (3), the light intensity is 750 lux, the light - dark cycle is L / D = 6 h / 18 h, and the temperature is 5 °C.

[0022] As a preferred technical solution, during the preservation step in step (3), gently shake the green bottle for 30 - 45 seconds every 72 hours.

[0023] Beneficial effects:

[0024] Compared with the prior art, the method for preserving Dunaliella salina strain with the preservation nutrient solution of Dunaliella salina and the green bottle in the present invention significantly prolongs the preservation time and survival rate of Dunaliella salina, reduces the cost of sub - culture preservation of Dunaliella salina, and can continuously and stably supply Dunaliella salina strains to the market. Description of the Drawings

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on the provided drawings.

[0026] Figure 1 For the Dunaliella salina figure at 12 months of preservation, with less accumulated carotenoids at this time;

[0027] Figure 2 For the Dunaliella salina figure at 18 months of preservation with more accumulated carotenoids, and the flagella are clearer. Detailed implementation manners

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0029] All raw materials used in the present invention are commercially available, and no specific limitation is made on their sources. The methods involved, unless otherwise specifically mentioned, are conventional methods and will not be elaborated one by one here. The preparation method of the magnetized disinfected seawater used in the present invention is that ordinary seawater is filtered through absorbent cotton, boiled, cooled, and then flows through a water magnetizer (magnetic induction intensity is 0.3T, flow rate is 3t / h, flow velocity is 0.2m / s). The beer used is commercially available yellow beer, and no limitation is made on its brand.

[0030] Example 1

[0031] The Dunaliella salina preservation nutrient solution includes the following raw materials in parts by weight: 90 parts of magnetized disinfected seawater, 3 parts of beer (original wort concentration of Tsingtao Beer is 8.0°P), 13 parts of NaCl, 0.03 parts of NH4NO3, 0.0004 parts of KH2PO4, 0.00005 parts of ferric citrate, 0.3 parts of NaHCO3, 0.04 parts of glycerol, 0.000003 parts of carramycin, and 0.0000009 parts of choline chloride; among them, NaCl is added in three equal amounts. The first addition is before inoculation, and the second and third additions are at 12 hours and 24 hours after inoculation respectively.

[0032] A method for preserving Dunaliella salina algal species with a green bottle at high salinity, using a green bottle body to preserve Dunaliella salina algal species, including the following steps:

[0033] (1) Add (300 mL) of the above-mentioned preservation nutrient solution to a green beer bottle (with a volume of 600 mL and a thickness of 1.5 mm), inoculate with Dunaliella salina algal seeds at a density of (10,000 cells / mL), gently shake well for 25 - 30 seconds, and then stopper the bottle mouth with a breathable sterile plug (a plug made of medical gauze wrapped around absorbent cotton).

[0034] (2) Store in a dark and low-temperature environment for 48 h, where the low temperature is 0 - 2 °C;

[0035] (3) Transfer to a light incubator for preservation. The light intensity is 600 lux, the light-dark cycle is L / D = 6 h / 18 h, and the temperature is 6 °C; gently shake the green bottle for 30 - 45 seconds every 72 hours.

[0036] Example 2

[0037] The preservation nutrient solution for Dunaliella salina includes the following raw materials in parts by weight: 92 parts of magnetized and disinfected seawater, 4 parts of beer (the original wort concentration of Tsingtao Beer is 8.0 °P), 13 parts of NaCl, 0.05 part of NH4NO3, 0.0005 part of KH2PO4, 0.00006 part of ferric citrate, 0.6 part of NaHCO3, 0.03 part of glycerol, 0.000005 part of kalamycin, and 0.0000007 part of choline chloride.

[0038] A method for preserving Dunaliella salina algal seeds in a green bottle with high salinity. The method uses a green bottle body to preserve Dunaliella salina algal seeds and includes the following steps:

[0039] (1) Add (250 mL) of the above-mentioned preservation nutrient solution to a green beer bottle (with a volume of 600 mL and a thickness of 1.5 mm), inoculate with Dunaliella salina algal seeds at a density of (20,000 cells / mL), gently shake well for 25 - 30 seconds, and then stopper the bottle mouth with a breathable sterile plug (a plug made of medical gauze wrapped around absorbent cotton).

[0040] (2) Store in a dark and low-temperature environment for 48 h, where the low temperature is 0 - 2 °C;

[0041] (3) Transfer to a light incubator for preservation. The light intensity is 750 lux, the light-dark cycle is L / D = 6 h / 18 h, and the temperature is 5 °C; gently shake the green bottle for 30 - 45 seconds every 72 hours.

[0042] Example 3

[0043] Dunaliella salina preservation nutrient solution, comprising the following raw materials in parts by weight: 95 parts of magnetized and disinfected seawater, 7 parts of beer (original wort concentration of Tsingtao Beer is 8.0°P), 15 parts of NaCl, 0.05 part of NH4NO3, 0.0008 part of KH2PO4, 0.00009 part of ferric citrate, 0.6 part of NaHCO3, 0.05 part of glycerol, 0.000005 part of kalamycin, and 0.0000006 part of choline chloride.

[0044] A method for preserving Dunaliella salina strains with a green bottle at high salinity, which uses a green bottle to preserve Dunaliella salina strains, comprising the following steps:

[0045] (1) Add (200 mL) of the above preservation nutrient solution to a green beer bottle (volume 600 mL, thickness 1.5 mm), inoculate Dunaliella salina strains at (30,000 cells / mL), gently shake well, the shaking time is 25 - 30 seconds, and then plug the bottle mouth with a breathable sterile plug (a plug made of medical gauze wrapped around absorbent cotton).

[0046] (2) Place it in a dark and low-temperature environment for 48 h, and the low temperature is 0 - 2°C;

[0047] (3) Transfer it to a light incubator for preservation, the light intensity is 900 lux, the light-dark cycle is L / D = 6 h / 18 h, and the temperature is 9°C; gently shake the green bottle for 30 - 45 seconds every 72 hours.

[0048] Comparative Example 1

[0049] The Dunaliella salina preservation nutrient solution does not include beer, and other components are the same as those in Example 2, and the preservation method is also the same as that in Example 2.

[0050] Comparative Example 2

[0051] In the Dunaliella salina preservation nutrient solution, NaCl is reduced to 5 parts, and other components are the same as those in Example 2, and the preservation method is also the same as that in Example 2.

[0052] Comparative Example 3

[0053] The Dunaliella salina preservation nutrient solution is the same as that in Example 2. In the preservation method, a white glass bottle (thickness 1.5 mm) is used, and other steps are the same as those in Example 2.

[0054] Comparative Example 4

[0055] The Dunaliella salina preservation nutrient solution is the same as that in Example 2. In the preservation method, step (2) of preserving at 0 - 2°C for 48 h is omitted, and other steps are the same as those in Example 2.

[0056] Comparative Example 5

[0057] The nutrient solution for preserving Dunaliella salina is the same as that in Example 2. In the preservation method, step (3) is to store at room temperature of 20 - 25°C, and other steps are the same as those in Example 2.

[0058] Comparative Example 6

[0059] The method of CN105886403A is adopted to preserve Dunaliella salina.

[0060] Comparative Example 7

[0061] Dunaliella salina is stored at a low temperature of 0°C - 4°C with a 15% SAM8 protective solution (a biological metabolic regulator with antioxidant effect). Refer to "Study on the Preservation Method of High - Concentration Nitzschia closterium minutissima" by Sun Jianhua et al. (1998, Acta Oceanologica Sinica, Vol. 20, No. 2).

[0062] Example 4

[0063] Experimental studies and statistical analyses were conducted on the survival of Dunaliella salina preserved in Examples 1 - 3 and Comparative Examples 1 - 7, and the results are shown in Table 1 below. (Data processing: The experiment was conducted 5 times. The SPSS software package was used for statistical analysis of the data. Taking Comparative Example 7 as the control group, a significant difference statistical analysis was carried out, with 0.05 as the significance level of difference.)

[0064] The PI - FDA staining method was used to measure the survival rate; the preservation cost includes water and electricity fees and drug fees, excluding labor costs.

[0065] Table 1 Survival rate

[0066]

[0067] Example 5

[0068] The cell components of the Dunaliella salina algal species preserved in Example 2 and Comparative Example 7 after 24 months were measured to investigate the impact of the method of the present invention on the cells. (Data processing in this example: The experiment was conducted 5 times. The SPSS software package was used for statistical analysis of the data. Taking Comparative Example 7 as the control group, a significant difference statistical analysis was carried out, with 0.05 as the significance level of difference.)

[0069] (1) Determination of fatty acids in algal cells

[0070] The algal liquid was centrifuged, the supernatant was discarded, and the algal mud was taken. After freeze - drying, 0.1 g of the freeze - dried sample was taken, saponified, methyl - esterified, extracted, and the fatty acid components were determined by a gas chromatograph (HP - 5890 type, Hewlett - Packard Company, USA). The mass fraction of fatty acid components was calculated by the peak area normalization method, and the results are shown in Table 2.

[0071] Table 2 Proportion of different types of fatty acids in total fatty acids

[0072]

[0073] It can be seen that the method of the present invention is beneficial to the increase of the fatty acid content, especially the polyunsaturated fatty acid content, in Dunaliella salina.

[0074] (2) Determination of algal cell protein

[0075] Take the preserved algal solution, centrifuge it at a speed of 6000 revolutions per minute for 3 minutes, rinse the algal sludge with distilled water, then centrifuge it again at a speed of 5000 revolutions per minute for 4 minutes, and repeat this process 3 times, and then freeze-dry it. Take 0.1 - 0.2 grams of the dried algal powder and determine the protein content by the Kjeldahl method.

[0076] (3) Determination of glycerol

[0077] The periodic acid oxidation method is adopted.

[0078] (4) Determination of β-carotene

[0079] 5 mL of algal solution → extraction with 90% acetone → supernatant → volume made up to 50 mL → measure OD with a 721 spectrophotometer 450 value

[0080] β-carotene content = (volume made up × 10 × OD 450 ) × 1000 ÷ (2500 × sampling volume)

[0081] In the formula, the volume unit is millilitre (mL).

[0082] The contents of algal cell protein, glycerol and β-carotene are shown in Table 3.

[0083] Table 3

[0084]

[0085] It can be seen from the test results of this example that the Dunaliella salina preserved by the method of the present invention has made remarkable technological progress in terms of cell quality.

[0086] In this specification, each example is described in a progressive manner. The key point of each example is to illustrate the differences from other examples. For the same or similar parts among the examples, reference can be made to each other.

[0087] The above description of the disclosed examples enables those skilled in the art to implement or use the present invention. Various modifications to these examples will be obvious to those skilled in the art. The general principles defined herein can be implemented in other examples without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these examples shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nutrient solution for preserving Dunaliella salina, characterized in that, It comprises the following raw materials in parts by weight: 90 - 95 parts of magnetized disinfected seawater, 3 - 7 parts of beer, 12 - 15 parts of NaCl, 0.03 - 0.07 parts of NH4NO3, 0.0004 - 0.0008 parts of KH2PO4, 0.00005 - 0.00009 parts of ferric citrate, 0.3 - 0.8 parts of NaHCO3, 0.02 - 0.05 parts of glycerol, 0.000003 - 0.000007 parts of kalamycin and 0.0000006 - 0.0000009 parts of choline chloride.

2. The nutrient solution for preserving Dunaliella salina according to claim 1, characterized in that, It comprises the following raw materials in parts by weight: 92 parts of magnetized disinfected seawater, 4 parts of beer, 13 parts of NaCl, 0.05 parts of NH4NO3, 0.0005 parts of KH2PO4, 0.00006 parts of ferric citrate, 0.6 parts of NaHCO3, 0.03 parts of glycerol, 0.000005 parts of kalamycin and 0.0000007 parts of choline chloride.

3. The nutrient solution for preserving Dunaliella salina according to claim 1 or 2, characterized in that, The addition of the NaCl is carried out in three equal amounts. The first addition is made before inoculation, and the second and third additions are made 12 hours and 24 hours after inoculation respectively.

4. A method for preserving Dunaliella salina algal species in a green bottle with high salinity, characterized in that, The steps for preserving the Dunaliella salina algal species in a green bottle body are as follows: (1) Add the preservation nutrient solution described in any one of claims 1 - 3 to the green bottle body, and inoculate the Dunaliella salina algal species. The addition amount of the preservation nutrient solution is 1 / 3 - 1 / 2 of the volume of the bottle body. (2) Preserve it in a dark and low - temperature environment for 48 h, and the low temperature is 0 - 2°C. (3) Transfer it to a light incubator for preservation. The light intensity is 600 - 900 lux, the light - dark cycle is L / D = 6 h / 18 h, and the temperature is 4 - 9°C.

5. The method according to claim 4, characterized in that, The thickness of the green bottle body ≥ 1.5 mm.

6. The method according to claim 5, characterized in that, The green bottle is a beer bottle.

7. The method according to claim 4, characterized in that, The step of inoculating the Dunaliella salina algal species in step (1) is as follows: Take pure Dunaliella salina algal species in the exponential growth phase for inoculation. The inoculation density is 10,000 - 30,000 cells / ml. Gently shake it well. The shaking time is 25 - 30 seconds, and then plug the bottle mouth with a breathable sterile plug.

8. The method according to claim 7, characterized in that, The breathable sterile plug is a plug made of medical gauze wrapped around absorbent cotton.

9. The method according to claim 4, characterized in that, In step (3), the light intensity is 750 lux, the light - dark cycle is L / D = 6 h / 18 h, and the temperature is 5°C.

10. The method according to claim 4, characterized in that, In the preservation link of step (3), gently shake the green bottle for 30 - 45 seconds every 72 hours.

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